Rab7 Pull-Down Activation Assay Kit
Cat. # 82501
Introduction
A. Background
Small GTPases are a super-family of cellular signaling regulators. The small Ras-like GTPase Rab7 is an evolutionary conserved protein that originally gained interest because of its capacity to revert the morphological phenotype of Ras-transformed fibroblasts. Rab7 is regulated by a large number of stimuli that include growth factors and cytokines, but also physical force and osmotic stress. Rab7 was shown to regulate multiple basic cellular processes. The best studied aspect of Rab7 function in endothelial cells involved its role in regulation of cell-cell junction formation and remodeling.
Rab7 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rab7-GTP, but not Rab7-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This assay provides the reliable results with consistent reproducibility.
The anti-Rab7-GTP monoclonal antibody can also be used to monitor the activation of Rab7 in cells and in tissues by immunohistochemistry.
B. Assay Principle
The Rab7 Activation Assay Kit uses configuration-specific anti-Rab7-GTP Mouse monoclonal antibody to measure Rab7-GTP levels in cell extracts or in vitro GTPγS loading Rab7 activation assays. Anti-Rab7-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rab7-GTP. Next, the GTP-bound Rab7 is pulled down by protein A/G agarose. Finally, the precipitated Rab7-GTP is detected through immunoblot analysis using Anti-Rab7 Rabbit Polyclonal Antibody.
C. Kit Compo
nents
1. Anti-Rab7-GTP Mouse Monoclonal Antibody (Cat. # 26923): One vial – 35 μL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rab7-GTP from all vertebrates.
2. Protein A/G Agarose (Cat. # 30301): One vial – 600 μL of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. # 30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Rab7 Rabbit Polyclonal Antibody (Cat. # 21069): One vial – 50 μL (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): One vial – 50 μl at 10 mM, use 5 μL of GTPγS for GTP-labeling of 0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304): One vial – 50 μl at 100 mM, use 5 μL of GDP for GDP-labeling of 0.5 mL of cell lysate.
7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 μL (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
D. Materials Needed but Not Supplied
1. Stimulated and non-stimulated cell lysates
2. Protease inhibitors
3. 4 °C tube rocker or shaker
4. 0.5 M EDTA at pH 8.0
5. 1.0 M MgCl2
6. 2X reducing SDS-PAGE sample buffer
7. Electrophoresis and immunoblotting systems
8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05% Tween-20)
9. Immunoblotting blocking buffer (TBST containing 5% Non-fat Dry Milk or 3% BSA)
10. ECL Detection Reagents
E. Example Results
The following figure demonstrates example results seen with the Rab7 Activation Assay Kit. For reference only.
Rab7 Activation Assay. Purified GST-tagged Rab7 proteins (Cat. #10134) were immunoprecipitated with the anti-Rab7-GTP mo
noclonal antibody (Cat. No. 26923) after treated with GDP (lane 1) or GTPγS (lane 2), and was blotted with anti-Rab7 polyclo
nal antibody (Cat. # 21069). Input co
ntrol is shown in the bottom panel.
Assay Procedure
A. Reagent Preparation
1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30301) briefly and dilute to 1X in deio
nized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 μg/mL leupeptin, or 10 μg/mL aprotinin.
B. Sample Preparation
Adherent Cells
1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the cells with activator or inhibitor as desired.
2. Aspirate the culture media and wash twice with ice-cold PBS.
3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cells (0.5-1 mL per 10 cm tissue culture plate).
4. Place the culture plates on ice for 10-20 minutes.
5. Detach the cells from the plates by scraping with a cell scraper.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.
Adherent Cells
1. Culture cells and stimulate with activator or inhibitor as desired.
2. Perform a cell count and then pellet the cells through centrifugation.
3. Aspirate the culture media and wash twice with ice-cold PBS.
4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place them on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27?-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.
C. In vitro GTPγS/GDP Protein for Positive and Negative co
ntrols
Note: In vivo stimulation of cells will activate approximately 10% of the available Rab7, whereas in vitro GTPγS protein loading will activate nearly 90% of Rab7.
1. Aliquot 0.5 mL of cell extract (or 1 μg of purified Rab7 protein) into two microcentrifuge tubes.
2. To each tube, add 20 μL of 0.5 M EDTA (final concentration of 20 mM).
3. Positive control: add 5 μL of 100 X GTPγS (Cat. # 30302) to the 1st tube
4. Negative control: add 5 μL of 100 X GDP (Cat. # 30304) to the 2nd tube.
5. Incubate both tubes at 30°C for 30 minutes with agitation.
6. Stop loading by placing the tubes on ice and adding 32.5 μL of 1 M MgCl2 (final concentration of 60 mM).
D. Affinity Precipitation of Activated G Protein
1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.
2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 μL anti-Rab7-GTP antibody (Cat. # 26923).
4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.
5. Quickly add 20 μL of resuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1 hour with gentle agitation.
7. Pellet the beads through centrifugation at 5,000 x g for 1 min.
8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet.
9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.
10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 μL of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5 minutes.
13. Centrifuge it at 5,000 x g for 10 seconds.
E. Western Blot Analysis
1. Load 15 μL/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).
2. Perform SDS-PAGE following the manufacturer’s instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s instructions.
Note: Steps 4-11 are at room temperature with agitation
4. Following electroblotting, immerse the PVDF membrane in 100% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used instead of PVDF, step 4 Should be skipped.
5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature with constant agitation.
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Incubate the membrane with Anti-Rab7 Rabbit Polyclonal Antibody (Cat. # 21069), which is freshly diluted 1: 50~500 (depending on the amount of Rab7 proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperature with constant agitation or at 4°C overnight.
8. Wash the blotted membrane three times with TBST, 5 minutes each time.
9. Incubate the membrane with a secondary antibody (Cat. # 29002), which is freshly diluted 1: 1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr at room temperature with constant agitation.
10. Wash the blotted membrane three times with TBST, 5 minutes each time.
11. Use the detection method of your choice such as ECL.
Rab7 活性檢測試劑盒/高質量試劑盒廠家Rab7 活性檢測試劑盒/高質量kit廠家
Rab7 活性檢測試劑盒/高質量kit廠家
Rab7 活性檢測試劑盒/高質量kit廠家 Rab7 活性檢測試劑盒/高質量試劑盒廠家
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